Rapid whole genome amplification

ABSTRACT

The present invention provides compositions and methods for rapidly amplifying target nucleic acid (e.g., using whole genome amplification) that allows small amounts of starting nucleic acid to be employed. In certain embodiments, the methods employ compositions that comprise: phi29 polymerase, exo- Klenow polymerase and/or Klenow polymerase, dNTPs, primers, and a buffering agent. In some embodiments, the target nucleic acid is amplified at a rate that would result in at least 1000-fold amplification in thirty minutes.

The present Application claims priority to U.S. Provisional ApplicationSer. No. 61/428,076 filed Dec. 29, 2010, the entirety of which isincorporated by reference herein.

FIELD OF THE INVENTION

The present invention provides compositions and methods for rapidlyamplifying target nucleic acid (e.g., using whole genome amplification)that allows small amounts of starting nucleic acid to be employed. Incertain embodiments, the methods employ compositions that comprise:phi29 polymerase, exo- Klenow polymerase and/or Klenow polymerase,dNTPs, primers, and a buffering agent. In some embodiments, the targetnucleic acid is amplified at a rate that would result in at least1000-fold amplification in thirty minutes.

BACKGROUND

In many fields of research such as genetic diagnosis, cancer research orforensic medicine, the scarcity of genomic DNA can be a severelylimiting factor on the type and quantity of genetic tests that can beperformed on a sample. One approach designed to overcome this problem iswhole genome amplification. The objective is to amplify a limited DNAsample in a non-specific manner in order to generate a new sample thatis indistinguishable from the original but with a higher DNAconcentration. The aim of a typical whole genome amplification techniqueis to amplify a sample up to a microgram level while respecting theoriginal sequence representation.

The first whole genome amplification methods were described in 1992, andwere based on the principles of the polymerase chain reaction. Zhang andcoworkers (Zhang, L., et al. Proc. Natl. Acad. Sci. USA, 1992, 89:5847-5851; herein incorporated by reference) developed the primerextension PCR technique (PEP) and Telenius and collaborators (Teleniuset al., Genomics. 1992, 13(3):718-25; herein incorporated by reference)designed the degenerate oligonucleotide-primed PCR method (DOP-PCR). PEPinvolves a high number of PCR cycles, generally using Taq polymerase and15 base random primers that anneal at a low stringency temperature.DOP-PCR is a method which generally uses Taq polymerase andsemi-degenerate oligonucleotides (such as CGACTCGAGNNNNNNATGTGG (SEQ IDNO: 12), for example, where N=A, T, C or G) that bind at a low annealingtemperature at approximately one million sites within the human genome.The first cycles are followed by a large number of cycles with a higherannealing temperature, allowing only for the amplification of thefragments that were tagged in the first step.

Multiple displacement amplification (MDA, also known as stranddisplacement amplification; SDA) is a non-PCR-based isothermal methodbased on the annealing of random hexamers to denatured DNA, followed bystrand-displacement synthesis at constant temperature (Blanco et al.,1989, J. Biol. Chem. 264:8935-40; Dean, F. B. et al. (2002)Comprehensive human genome amplification using multiple displacementamplification; Proc. Natl. Acad. Sci. USA 99,5261; and Van, J. et al.(2004) Assessment of multiple displacement amplification in molecularepidemiology. Biotechniques 37, 136; all of which are hereinincorporated by reference). It has been applied to small genomic DNAsamples, leading to the synthesis of high molecular weight DNA withlimited sequence representation bias (Lizardi et al., Nature Genetics1998, 19, 225-232; Dean et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99,5261-5266; both of which are herein incorporated by reference). As DNAis synthesized by strand displacement, a gradually increasing number ofpriming events occur, forming a network of hyper-branched DNAstructures. The reaction can be catalyzed by the Phi29 DNA polymerase orby the large fragment of the Bst DNA polymerase. The Phi29 DNApolymerase possesses a proofreading activity resulting in error rates100 times lower than the Taq polymerase. MDA type methods, however,require many hours (e.g., 6 hours) to generate a sufficient foldamplification.

What is needed are whole genome amplification methods that are fasterthan known methods.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for rapidlyamplifying target nucleic acid (e.g., using whole genome amplification)that allows small amounts of starting nucleic acid to be employed. Incertain embodiments, the methods employ compositions that comprise:phi29 polymerase (or Bst DNA polymerase, or other strand displacingpolymerase), exo- Klenow polymerase and/or Klenow polymerase (or otherDNA polymerase), dNTPs, primers, and a buffering agent. In someembodiments, the target nucleic acid is amplified at a rate that wouldresult in at least 1000-fold amplification in thirty minutes.

In some embodiments, the present invention provides methods ofamplifying target nucleic acid comprising: a) providing a samplecomprising: i) target nucleic acid, ii) phi29 polymerase (or Bst DNApolymerase, or other strand displacing polymerase), iii) exo- Klenowpolymerase and/or Klenow polymerase (or other DNA polymerase), iv)dNTPs, v) primers, and vi) a buffering agent; and b) treating the sampleunder conditions such that the target nucleic acid is amplified therebygenerating amplified target nucleic acid. In certain embodiments, thetreating is conducted for no more than 4 hours (e.g., no more than 2hours . . . no more than 1.5 hours . . . no more than 1.0 hour . . . nomore than 45 minutes . . . no more than 35 minutes . . . no more than 30minutes . . . no more than 15 minutes). In certain embodiments, thetreating is conducted between 10 minutes and 2 hours (e.g., 15minutes—1. 5 hours or 20 minutes to 1 hour).

In certain embodiments, the present invention provides compositions orsamples comprising: i) phi29 polymerase (or Bst DNA polymerase, or otherpolymerase, such as a strand displacing polymerase), ii) exo- Klenowpolymerase and/or Klenow polymerase (or other DNA polymerase), iii)dNTPs, iv) primers, and iv) a buffering agent. In particularembodiments, the compositions or samples further comprise target nucleicacid.

In particular embodiments, the treating is under isothermal conditions.In other embodiments, the target nucleic acid is amplified at a ratethat would result in at least 200-fold - . . . 500-fold . . . 1000-fold. . . 1500-fold . . . 2000-fold . . . or at least 2500-foldamplification in 30 minutes. In certain embodiments, the target nucleicacid is present in the sample (or composition) at a level between 1 ngand 100 ng (e.g., 1-10 ng; 10-40 ng; 50-75 ng; 75-100 ng).

In other embodiments, the primers are random primers. In certainembodiments, the target nucleic acid is genomic DNA. In someembodiments, the sample further comprises a phosphatase. In particularembodiments, the sample further comprises a pyrophosphatase. Inadditional embodiments, the dNTPs are at a concentration of at least 10mM of each of the four bases (e.g., at least 10 mM . . . at least 15 mM. . . at least 20 mM . . . at least 25 mM . . . at least 30 mM orhigher).

In some embodiments, the sample comprises the Klenow polymerase. Infurther embodiments, the sample comprises the exo- Klenow polymerase. Inadditional embodiments, the buffering agent comprisestris(hydroxymethyl) aminomethane (TRIS).

In particular embodiments, the sample (or composition) further comprisesat least one component (or at least two, or at least three, or at leastfour, or all five components) selected from the group consisting of: anemulsifier, a divalent metal cation, an inorganic salt, an alpha-linkeddisaccharide, and a reducing agent. In other embodiments, the emulsifieris a polysorbate. In some embodiments, the polysorbate is selected fromthe group consisting of: Tween 20, Tween 40, Tween 60, or Tween 80. Inother embodiments, the inorganic salt is ammonium sulfate. In additionalembodiments, the alpha-linked disaccharide comprises Trehalose. In someembodiments, the reducing agent comprises dithiothreitol (DTT).

DESCRIPTION OF THE FIGURES

FIG. 1 shows fold-amplification WGA results from Example 1 where the A7buffer and B4 enzyme were tested at various times (15 minutes and 30minutes) and various temperatures (30 C, 37 C, and 39 C).

FIG. 2 shows a comparison of the fold-amplification results using the A7and A9 buffers in whole genome amplification at 37 C for 30 minutes.

FIG. 3 shows fold-amplification WGA results from Example 3 where variousenzymes were tested.

FIG. 4 shows fold-amplification WGA results from Example 4 where variousenzyme and enzyme combinations were tested.

FIG. 5 shows fold-amplification WGA results from Example 5 where variousconditions and enzymes were tested in A9 buffer.

FIG. 6 shows fold-amplification WGA results from Example 6 where variousamounts of Klenow exo- were tested in A9 buffer.

FIG. 7 shows fold-amplification WGA results from Example 7 where variousenzymes were tested in A9 buffer.

FIG. 8 shows fold-amplification WGA results from Example 8 where variousconcentrations of dNTPs were tested.

FIG. 9 shows a comparison of the WGA fold-amplification results usingthe A7 buffer (with B4 enzyme mix) and the A9 buffer (using the B5enzyme mix) as described in Example 9.

FIG. 10 shows a comparison of the WGA fold-amplification betweenstandard conditions (using 6 hours) and various combinations of Phi29,Klenow or Klenow exo-, and pyrophosphatase (using 40 minutes).

DETAILED DESCRIPTION

The present invention provides compositions and methods for rapidlyamplifying target nucleic acid (e.g., using whole genome amplification)that allows small amounts of starting nucleic acid to be employed. Incertain embodiments, the methods employ compositions that comprise:phi29 polymerase, exo- Klenow polymerase and/or Klenow polymerase,dNTPs, primers, and a buffering agent. In some embodiments, the targetnucleic acid is amplified at a rate that would result in at least1000-fold amplification in thirty minutes.

The present invention provides methods for the rapid amplification ofwhole genomic DNA. In certain embodiments, the methods of the presentinvention are able to generate significant amounts of DNA from tracelevels. This allows samples with very low levels of DNA content to beamplified and used, for example, in further diagnostic tests (e.g.,forensic testing). For example, the reaction conditions provided byembodiments of the present invention generate 2-5 ug of DNA from 1 ng ofstarting DNA (˜2500× amplification), in a 30 minute reaction.

In the prior art, whole genome amplification reactions took many hours(e.g., 6-12 hours) to generate a significant amount of material. Thepresent invention greatly shortens this time requirement (e.g., reducesthe time to 30 minutes).

In certain embodiments, the buffer and enzyme mixture shown in Table 1,or similar mixtures are employed.

TABLE 1 Template DNA Tris HCL 0.04025 M Tris Base 0.00975 M MgCl2 0.012M (NH₄)₂SO₄ 0.01 M Trehalose 0.5656 M Tween-40 1% DNTP mix 100 mM (25 mMeach) Bioline 2.8 mM DTT 0.004 M Primer Mix 0.05 mM Enzyme mix 1 ul = 50U Phi29 & 20 U Klenow & 009 U 100 ul Pyrophosphatase 2 ul rxnOne exemplary protocol using the mixture from Table 1 is as follows. Thestarting genomic DNA (>10 pg-1 ng) is added to the buffer mixture inTable 1 to a final volume of 96 ul. The DNA is heat denatured at 95 Cfor 1 minute, and the cooled to 4 C and held at that temperature for 30minutes (for amplification). Then mixture is then incubated at 75 C for10 minutes for heat inactivation of the enzymes. The levels ofamplification may be determined qualitatively by gel electrophoresis andquantitatively by qPCR reactions.

The present invention shortens the time and increases the yield ofcurrent whole genome amplification protocols. Therefore, in someembodiments, it is useful in any assay that requires large amounts ofDNA or assays that require a trace sample to be split into many testreactions such as Next Gen Sequencing Sanger sequencing, DNAmicroarrays, Broad-range PCR.

EXAMPLES Materials and Methods for the Examples Below

The buffer and enzyme formulations that were employed are shown in Table2 below:

TABLE 2 Buffer Formulations A7 base Pellet A-Mix A9 base Tris pH 7.6 50mM 50 mM 50 mM MgCl2 12 mM 12 mM 12 mM (NH4)2SO4 10 mM 10 mM 10 mMBetaine 566 mM 0 mM 0 mM Trehalose 566 mM 566 mM 566 mM Tween 40 1%(w/v) 0.05% (w/v) 1% (w/v) DTT 0 mM 4 mM 0 mMIt is noted that making complete A7 buffer or A9 buffer involves addingprimers, dNTPs and DTT as described in the Examples below.

Pellet A-mix already has primers and DNTPs at the concentrations foundin A7 The additional enzyme mixes and enzyme pellets employed are shownin Table 3 below.

TABLE 3 Enzyme Pellets B4 (ie #11, B4, etc.) Phi 29 Polymerase 44.8units 44.8 units Pyrophospatase 0.007 units 0.007 units Polymerase Pol I0.90 units 0.90 units BSA 37.5 ug 37.5 ug Glycerol 50% (v/v) 0% (v/v)It is noted that all of the lyophilized enzymes used are the sameformulation despite different names.

A-mix for pellets Per 100 ul Rxn A mix 47.80 DTT 0.38 Primer 4.77 DNTP1.91 Poly A 0.14 total 55.00 water 15.00

A-mix fix for pellets Per 100 ul Rxn A mix 53.13 DTT 0.38 Primer 4.77DNTP 1.91 Poly A 0.14 total 60.33 water 9.68

Example 1 Selection of Time and Temperature for Whole GenomeAmplification Reactions

In this Example, varying temperature (30, 37 and 39 C) were tested for15 and 30 minutes with whole genome amplification methods. Each reactionhas 100 ul total volume in A7 complete with 1 ng human genomic DNA. Thereactions were heated to 95 C for 1 minute and then cooled to 0 C andheld at that temperature for 5 minutes. Then, 5 ul of B4 whole genomeamplification (WGA) enzymes were added, the reactions were mixed withvortexing and held at 4 C for 10 m, followed by shifting the reactionsto either 30 C, 37 C or 39 C for either 15 or 30 minutes. The reactionswere heat killed at 65 C for 10 minutes followed by holding thereactions at 4 C till needed. The results are shown in FIG. 1, whichshowed that 30 minutes at 37 C gave good fold amplification.

It is noted, to make A7 complete, 1000 ul of A7 base (see Table 2) wascombined with 6.1 ul 1M DTT, 75.9 ul of 1 mM random 7 mers, 30.4 ul ofdNTP (25 mM each) and 2.3 ul of polyA (1 mg/ml sonicated). Forquantitating the results (shown in FIG. 1), 2 ul of each reaction wasdiluted in 18 ul of DNA dilution buffer, sonicated in a water bathsonicator >200 W for 3 minutes at 4 C. The samples were then tested inqPCR reaction with a standard curve of human genomic DNA.

Example 2 Whole Genome Amplification Buffer Optimizations

This Example performed whole genome amplification at 37 C for 30 minuteswith different buffer formations. All reactions were at 100 ul totalvolume in thin walled PCR tubes with 1 ng of B. anthracis (BA) DNA. Thebuffer in reaction 1 was “Amix for pellets” buffer. Reaction 2 bufferwas “Amix-fix for pellets,” reaction 3 buffer was A7 complete, andreaction buffer #4 was A9-pellets+H20.

All reactions were heated to 95 C for 2 minutes, then cooled to 4 C for5 minutes. To reactions 1, 2 and 4 there was 1 WGA enzyme pellet #11added. B4 enzyme mix (5 ul) was added to reaction 3. All reactions werevortexed, heated to 37 C for 30 minutes, followed by 65 C for 10 m andshifted to 4 C to hold. No template controls were included with no BADNA. Each test condition was tested in duplicate. Fold amplification wasdetermined with BA specific qPCR reactions. The results are shown inFIG. 2, which show that the A9 buffer, using 37 C for 30 minutes,provided increased fold amplification

Example 3 WGA Enzyme Optimization

This Example explored the effects of additions of E. coli DNApolymerase, Klenow exo- enzyme, and T4 DNA polymerase to the WGAreactions with and without the addition of higher levels of dNTP.

A9 complete buffer was made with the following reagents:

A9 base bag5 1000 DTT 1M 6.5 Primers  1 mM 81.2 dNTP 25 mM each (C13)32.5 dH20 16.2 1136.4 total volume.All reactions were at 100 ul total volume in A9 complete buffer in thinwalled PCR tubes with 200 pg of K. pneumoniae (KP) DNA. Each reactionwas heated to 95 C for 1 m and cooled to 4 C for 5 m. Then the followingadditions were made:

To reaction 1, 1 WGA enzyme bead #11.

To reaction 2, 1 WGA enzyme bead #11 and 40 u of Klenow exo-.

To reaction 3, 1 WGA enzyme bead #11, 40 u of Klenow exo- and 4 ul dNTPmix (10 mM each).

To reaction 4, 1 WGA enzyme bead #11 and 6 u of T4 DNA polymerase.

To reaction 5, 1 WGA enzyme bead #11 and 18 u of T4 DNA polymerase.

To reaction 6, 5 ul of B4 enzyme mix.

To reaction 7, 5 ul of B4 enzyme mix and 40 u of Klenow exo-.

To reaction 8, 5 ul of B4 enzyme mix and 40 u of Klenow exo- and 4 ul ofdNTP.

All reactions were brought to 100 ul with H20. Each reaction wasvortexed well and incubated at 37 C for 30 m, followed by a 75 C 10 mincubation and then the reactions were stored at −20 C. The DNAamplification was quantitated with a K.pneumoniae specific qPCRreaction. Results are shown in FIG. 3, which shows that additionalKlenow exo- and dNTPs enhances the speed of WGA, while T4 DNA polymerasedecreases the speed of WGA.

Example 4 Addition of Other DNA Polymerases to WGA Reaction

This Example was used to determine the effects of the addition of otherDNA polymerases to the WGA reaction. Each reaction was run in A9complete with 1 ng of K.pneumoniae genomic DNA. Each reaction contained70 ul and was heated to 95 C for 1 m followed by 15 minutes at 4 C.

To reaction 1 was added 50 u of Phi29 DNA polymerase.

To reaction 2 was added 50 u of Phi29 DNA polymerase and 5 u of E.coliDNA polymerase.

To reaction 3 was added 50 u of Phi29 DNA polymerase, 5 u of E.coli DNApolymerase and 40 u of Klenow exo-.

To reaction 4 was added 5 u of E.coli DNA polymerase and 40 u of Klenowexo-.

To reaction 5 was added 40 u of Klenow exo-.

To reaction 6 was added 5 ul of B4 WGA enzyme mix.

Each reaction was incubated at 37 C for 30 m followed by a heatinactivation at 75 C for 10 m. Reactions were stored at −20 C. Theresults are shown in FIG. 4.

Example 5 Optimizing WGA Reaction Components in A9 Complete Buffer

All reactions were set up to be in a 100 ul total volume. For eachreaction, 70 ul A9 complete was combined with 1 ul (200 pg/ul)K.pneumoniae genomic DNA and 19 ul of H20. This was heated to 95 C for 2minutes, and then cooled to 4 C and held at 4 C for 10 m. Reaction 1 isthe control reaction, to which was added 5 ul of H20. Reaction 2received 4 ul of H20 and 1 ul of phi29 DNA polymerase (100 u/ul).Reaction 3 received 4 ul of H20 and 1 ul of dNTP (10 mM each). Reaction4 received 4 ul of H20 and 1 ul of E.coli DNA polymerase Klenow fragmentexo- (40 u/ul). Reaction 5 received 5 ul of dNTP. To all reactions wasadded 1 bead of lyophilized enzyme B4. The reactions were mixed well andincubated at 37 C for 30 minutes, followed by 65 C for 10 m and thenstored at −20 C overnight. The quantization was done with qPCR specificfor K. pneumoniae DNA. The results are shown in FIG. 5.

Example 6 WGA Enhancement with Klenow exo-

In this Example, all reactions were set up in 100 ul total volume withA9 complete buffer. Each reaction contained 70 ul of A9 buffer, 1 ul ofK. pneumoniae DNA (200 pg/ul), and 21 ul of H20. All reactions wereheated to 95 C for 1 m and then held at 4 C for 5 m.

To reaction 2, 6 u of Klenow DNA polymerase.

To reaction 3, 18 u of Klenow.

To reaction 4, 6 u of Klenow exo-.

To reaction 5, 18 u of Klenow exo-.

To reaction 6, 40 u of Klenow exo-.

All reactions were brought to 95 ul with H20. To all reactions was added1 WGA enzyme pellet. The reactions were vortexed and heated at 37 C for30 minutes, followed by 65 C for 10 m and stored at −20 C. Thequantitation was performed with qPCR specific for Kp DNA. The resultsare shown in FIG. 6, which shows that 40 u of Klenow exo- enhances fastWGA, while Klenow did not in this particular Example.

Example 7 WGA Enzyme Optimization Phi29 Titration +/− Klenow exo-

In this Example, all reactions were at 100 ul volume in thin walled PCRtubes, with A9 complete buffer and 1 ng of Kp DNA. All reactions wereheated to 95 C for 1 m and then held at 4 C for 5 m. Reaction 1 received25 u of phi29 DNA polymerase, reaction 2 got 50 u, reaction 3 got 100 u,reaction 4 received 25 u, reaction 5 received 50 u and reaction 6received 100 u of phi29DNA polymerase. In addition, reaction 4-6received 40 u of Klenow exo-.

The reactions were incubated at 37 C for 30 minutes, followed by 75 Cfor 10 m and then held at 4 C till required. Quantitation was with KPspecific qPCR reactions. FIG. 7 shows the results, which shows that 100u phi29+40 u Klenow exo- works well for fast WGA (giving over 2,500 foldamplification in 30 minutes).

Example 8 WGA dNTP Optimization

In this Example, each reaction was 100 ul in thin walled PCR tubes withA9 complete buffer with 1 ng of Kp DNA. All reactions were heated to 95C for 1 minute and then held at 4 C for 5 minutes. Reaction 1-5 received100 u of phi29 DNA polymerase. In addition, reaction 2-5 received 40 uof Klenow exo- enzyme. Also, reaction 3-5 received additional dNTP.Reaction 3 received a 400 uM increase in dNTP, reaction 4 received a 200uM increase and reaction 5 received a 100 uM increase. All reactionswere vortexed well, and incubated at 37 C for 30 minutes, followed by 75C for 10 minutes and then held at 4 C. Quantitation was with KP specificqPCR reactions. FIG. 8 shows the results and shows that additional dNTPfurther enhances the fold amplification for WGA.

Example 9 Exemplary WGA Conditions

In this Example, all reactions were at 100 ul in thin walled PCR tubes.Reaction 1 contained buffer A7 and the B4 enzyme cocktail. Reaction 2contained buffer A9 and 100 u of phi29 DNA polymerase and 40 u of Klenowexo- and a 200 uM increase in dNTP. Both reactions used 1 ng ofK.pneumoniae DNA as starting material. Prior to the enzyme addition, thereactions were heated to 95 C for 1 minute followed by 4 C for 5minutes. Then the appropriate enzyme cocktails were added and thereactions were mixed well.

Reaction 1 was incubated at 30 C for 30 minutes, reaction 2 wasincubated at 37 C for 30 minutes. Then both reactions were heated to 75C for 10 minutes and held at 4 C. Quantitation was with KP specific qPCRreactions. FIG. 9 shows the results that shows that the A9 buffer and B5enzyme mix (B5 enzyme mix—100 u phi29 DNA pol, 40 u Klenow exo-, 20 mMdNTP) provided dramatically superior results, with over 2500-foldamplification in 30 minutes by WGA.

Example 10 Exemplary Fast WGA Conditions

This Example compares the standard A7 buffer to the A9 buffer of thepresent invention with additional dNTPs. All reactions were at 100 ultotal volume and contained 1 ng of Kp DNA template. All reactions wereheated to 95 C for 1 minute and then held at 4 C for 5 minutes prior toenzyme addition. All reaction conditions were tested in triplicate.

The standard WGA reaction (reaction #1) contained A7 complete buffer andB4 enzyme cocktail. The reaction was for 6 hours at 30 C, followed by 65C for 10 minutes. Reaction #2 was with A9 buffer, 100 u phi29 DNApolymerase and 40 u of Klenow exo- DNA polymerase. Reaction #3 wasidentical to reaction #2 with the addition of 0.02 u of pyrophosphatase.Reaction #4 was with A9 buffer, 100 u phi29 DNA polymerase and 50 u ofKlenow DNA polymerase. Reaction #5 was identical to reaction #4 with theaddition of 0.02 u of pyrophosphatase. Reactions 2-5 were incubated at37 C for 40 m, then 75 C for 10 m. FIG. 10 shows the results.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

1. A method of amplifying target nucleic acid comprising: a) providing asample comprising: i) target nucleic acid, ii) phi29 polymerase, iii)exo- Klenow polymerase and/or Klenow polymerase, iv) dNTPs, v) primers,and vi) a buffering agent; and b) treating said sample under conditionssuch that said target nucleic acid is amplified thereby generatingamplified target nucleic acid.
 2. The method of claim 1, wherein saidtreating is under isothermal conditions.
 3. The method of claim 1,wherein said target nucleic acid is amplified at a rate that wouldresult in at least 500-fold amplification in 30 minutes.
 4. The methodof claim 1, wherein said target nucleic acid is amplified at a rate thatwould result in at least 2000-fold amplification in 30 minutes.
 5. Themethod of claim 1, wherein said target nucleic acid is present in saidsample at a level between 1 ng and 100 ng.
 6. The method of claim 1,wherein said primers are random primers.
 7. The method of claim 1,wherein said target nucleic acid is genomic DNA.
 8. The method of claim1, wherein said sample further comprises a phosphatase.
 9. The method ofclaim 1, wherein said sample further comprises a pyrophosphatase. 10.The method of claim 1, wherein said dNTPs are at a concentration of atleast 10 mM of each of the four bases.
 11. The method of claim 1,wherein said sample further comprises at least one component selectedfrom the group consisting of: an emulsifier, a divalent metal cation, aninorganic salt, an alpha-linked disaccharide, and a reducing agent. 12.A composition comprising: i) phi29 polymerase, ii) exo- Klenowpolymerase and/or Klenow polymerase, iii) dNTPs, iv) primers, and iv) abuffering agent.
 13. The composition of claim 12, further comprisingtarget nucleic acid.
 14. The composition of claim 13, wherein saidtarget nucleic acid is present in said sample at a level between 1 ngand 100 ng.
 15. The composition of claim 12, wherein said primers arerandom primers.
 16. The composition of claim 12, wherein said samplefurther comprises a phosphatase.
 17. The composition of claim 12,wherein said sample further comprises a pyrophosphatase.
 18. Thecomposition of claim 12, wherein said dNTPs are at a concentration of atleast 10 mM of each of the four bases.
 19. The composition of claim 12,wherein said sample further comprises at least one component selectedfrom the group consisting of: an emulsifier, a divalent metal cation, aninorganic salt, an alpha-linked disaccharide, and a reducing agent. 20.The composition of claim 19, wherein said emulsifier is a polysorbate.